The full length cDNA cloning and expression analysis of EcR from the Chinese mitten crab(Eriocheir sinensis)

To study the regulatory role of ecdysteroid receptor(EcR)in molting and ovarian development of Chinese mitten crab,the full-length EcR gene of Eriocheir sinensis was cloned by using reverse transcriptase polymerase chain reaction(RT-PCR)and rapid-amplification of cDNA ends(RACE).The full-length cDNA sequence of EcR was 2 156 bp,and included a 1269bp ORF which encoded 422 amino acid residues.The alignment of EcR amino acid sequence of Eriocheir sinensis shared 89% identity with Uca pugilator.Quantitative real-time PCR(qRT-PCR)was used to quantify the relative expression level of EcR in different tissues,molting process,ovarian process and regulation of MF on ovarian EcR in E.sinensis The result showed the EcR mRNA were expressed in all tissues examined and highly in Y-organ,with small amount in ovary and muscle,and trace in hepatopancreas,gill,heart,stomach,intestine,thoracic ganglion and cerebral ganglion.During the molting process,the expression levels of EcR mRNA of Y-organ remained low from AB period to C period,then significantly increased in D period and E period.(P<0.05);the amount of EcR expression in hepatopancreas was rising from AB period to D period,and decreased in E period;EcR expression in muscle was highest in AB period(P<0.05);EcR expression in gill was highest in D period(P<0.05).In the process of Eriocheir sinensis ovarian development,the levels of EcR expression in ovary gradually increased to the maximum from stage Ⅰ to Ⅳ,low in stage Ⅴ.In injection experiment,EcR expression of the MF1(1 μg/crab)group had no significant difference with the control group and physiological saline group,MF2(2 μg/crab)group(P<0.05)had a marked increase;In vitro experiment,EcR expression was significantly increased in MF1(10^-8 mol/L)group and MF2(10^-7 mol/L)group(P<0.05).The results indicated that EcR might play an important role during molting process and ovarian development in Eriocheir sinensis,and MF could regulate EcR gene expression in ovarian.However,further studies are required to explore the underlying molecular mechanisms and clarify the function of EcR mRNA.